Discovery of RMC-5552, a Selective Bi-Steric Inhibitor of mTORC1, for the Treatment of mTORC1-Activated Tumors

Hyperactivation of mTOR kinase by mutations in the PI3K/mTOR pathway or by crosstalk with other mutant cancer drivers, such as RAS, is a feature of many tumors. Multiple allosteric inhibitors of mTORC1 and orthosteric dual inhibitors of mTORC1 and mTORC2 have been developed as anticancer drugs, but their clinical utility has been limited. To address these limitations, we have developed a novel class of “bi-steric inhibitors” that interact with both the orthosteric and the allosteric binding sites in order to deepen the inhibition of mTORC1 while also preserving selectivity for mTORC1 over mTORC2. In this report, we describe the discovery and preclinical profile of the development candidate RMC-5552 and the in vivo preclinical tool compound RMC-6272. We also present evidence that selective inhibition of mTORC1 in combination with covalent inhibition of KRASG12C shows increased antitumor activity in a preclinical model of KRASG12C mutant NSCLC that exhibits resistance to KRASG12C inhibitor monotherapy.

All proteins were then further purified by size-exclusion chromatography using a SuperDex-75 (Cytiva) column equilibrated with PBS (pH 7.4). Proteins were concentrated to 10 mg/mL.
Expression and purification of mTORC1 complex. mTORC1 complex (mTOR-mLST8-RAPTOR) was expressed and purified as previously described. 3 Briefly, an HEK293-F cell line was stably transfected with pcDNA3.1 vectors for FLAG tagged mTOR, mLST8, and RAPTOR.
Complex was affinity purified from cell lysate via anti-FLAG agarose beads and the tag was removed by incubation with TEV protease. Additional nickel and anti-FLAG columns removed the protease and cleaved tags, respectively.
The complex was further purified by size-exclusion chromatography using a  (Cytiva) column equilibrated with 20 mM Tris pH 8.0, 500 mM NaCl, 10 mM DTT, and 10% glycerol and then concentrated to 4.4 mg/mL.

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Crystallization, X-ray data collection, and crystal structure determination. Crystals of the FKBP12-FRB-11 and FKBP12-FRB-12 ternary complexes were obtained using previously described methods. 4  plates were stored at 293 K and crystals were harvested after 2 weeks. Crystals were cryoprotected with mother liquor supplemented with 20% glycerol and vitrified by plunging into liquid nitrogen. X-ray diffraction data have been collected at the SWISS LIGHT SOURCE (SLS, Villigen, Switzerland) at 100 K. The crystals belong to space group P 64 2 2.
Data were processed using XDS and XSCALE.
The phase information necessary to determine and analyze the structure was obtained by molecular replacement using a previously obtained crystal structure. Subsequent model building and refinement was performed according to standard protocols with the software packages CCP4 (REFMAC5 and COOT). The ligand parameterization and generation of the corresponding library files were carried out with CORINA. Statistics of the final structure and the refinement process are listed in Table S6 and Table S7.
Cryo-EM sample and grid preparation. Cryo-EM samples were prepared using a crosslinking procedure previously described. 5  Cryo-EM data collection, processing, and structure refinement. Cryo-EM data were collected on a 300kV Titan Krios G3i microscope equipped with a K2 Summit direct electron detector (Gatan) and GIF Quantum energy filter (20 eV slit width) using SerialEM 3.8.6. 6 12,808 movies were collected with a pixel size of 0.55 Å in super resolution mode at the specimen level, defocus range of -1.0 to -3.0 μm and a total dose of 48 electrons/Å2 fractionated to 40 frames.
Data processing was carried out with Relion3.1. 7 Motion correction was performed using Motioncor2 8 with a binning factor of 2, generating micrographs at a pixel size of 1.1 Å. Contrast transfer function (CTF) parameters were estimated by Kai Zhang's GCTF. 9 10,649 adequate micrographs without severe ice contaminations were used for subsequent data processing. S13 1,757,905 particles were semi-automatically picked using a Laplacian-of-Gaussian filter and subject to multiple rounds of 2D/3D classifications to remove poor quality particles. After screening, 805,027 particles were retained for 3D refinement without imposed symmetry, on which CTF refinement and Bayesian particle polishing were performed. 3D refinement with polished particles and C2 symmetry yielded a 3.1 Å reconstruction according to the FSC=0.143 criterion after postprocessing with a soft mask. To further improve the resolution of monomeric form, we computationally split each particle to two monomers by subtracting their signal with a monomer mask, resulting in 1,610,054 pseudo-monomer particles. 3D refinement with these pseudo-monomer particles yielded a 2.9 Å map after postprocessing. Local resolution was estimated by Relion's built-in implementation. The final map was sharpened anisotropically using Phenix LocalAnisoSharpen.
The mixture was extracted with EtOAc (2 x 100 mL), and the organic phases were washed with H2O (75 mL), sat. NaHCO3 (75 mL), and brine (75 mL To a solution of 12 (4.50 g, 4.84 mmol, 1.0 equiv) in DCM (180 mL) was added powdered 4Å molecular sieves (6.0 g). The mixture was stirred at room temperature for 1 h and then pyridine (3.91 mL, 48.4 mmol, 10 equiv) was added. The mixture was cooled to -10 °C and 4-nitrophenylchloroformate (0.990 g, 4.91 mmol, 1.0 equiv) was added in one portion. The reaction was allowed to slowly warm to room temperature and after 3 h the reaction mixture was cooled to 0 °C and 4-nitrophenylchloroformate (250 mg, 1.24 mmol, 0.3 equiv) was added. The mixture was warmed to room temperature and after 1 h the reaction mixture was filtered through a pad of celite and the pad was washed with DCM (140 mL). The filtrate was washed with H2O (120 mL) and sat NaHCO3 (2 x 120 mL). The organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude material was purified by flash chromatography (20→50% EtOAc/hex) to yield a white stiff foam. The material was taken up in MeCN during which time a white solid formed. The solid was filtered, washed with additional MeCN and allowed to air dry to afford the desired product (4.51 g, 85% yield) as an off-white stiff foam.